A fundamental question in male reproductive biology is the function of estrogen in the male. Our data and others have shown estrogen receptors(ER) to be concentrated in the efferent ductule and initial segment epididymidis. Despite this important information, there is little known regarding the role of estrogen in the male, even though efferent ductule occupy one-third of the caput epididymidis in humans. Our preliminary data using the ER-alpha knockout mouse (ERKO) and antiestrogen treatment of normal mice clearly indicate that estrogen regulates fluid and ion reabsorption in the efferent ductule. This is the first report of an essential function for estrogen in the male reproductive tract. Based on these and other preliminary data, we propose an overall hypothesis that estrogen regulates fluid reabsorption in efferent ductule through direct or indirect alterations in ion transport. This hypothesis will be tested using the following specific aims: 1. Determine the effects of antiestrogen treatment on the concentration of sperm in the epididymis and fertility in mice. 2. Measure the magnitude of fluid reabsorption regulated by estrogen in efferent ductules. 3. Identify alteration in the electroneutral NaCl absorptive mechanism induced in efferent ductule epithelium by antiestrogen treatment or transgenic estrogen receptor disruption. 4. Identify the role of estrogen in the regulation of gene expression for proteins involved in the ion transport processes found in efferent ductule epithelium. We will test the hypothesis that ER dysfunction will inhibit the ability of efferent ductule to reabsorb luminal fluid and thus the ability to move sperm into the epididymis in an environment of normal concentration required for sperm maturation. Both in vivo and in vitro experiments will be used to provide physiological relevance to these studies. To understand the physiological mechanisms, fluid movement in the ductule will be measured using microcannulation and micropuncture. Ultrastructural analysis of the endocytotic apparatus will be used to determine effects of antiestrogens on the ability of epithelial cells to take up electron-dense particles from the lumen. The function of ion transport proteins will be examined in vitro by voltage-clamped electrophysiology. Finally, we will determine if the effects of estrogen on fluid reabsorption are mediated by the regulation of gene expression in the efferent ductule. We have established primary epithelial cell cultures of ductuli efferentes that express ER and maintain electrophysiological properties consistent with a polarized epithelium. This research will provide basic information that will allow us to study disease processes that target the head of the epididymis and lead to male infertility.